Recombinant production of pharmaceutical proteins is pivotal, not only for personalized medicine. While most biopharmaceuticals are produced in mammalian cell culture, plant-made pharmaceuticals (PMP) are gaining momentum with the first product released to the market (protalix.com). Although posttranslational modifications (PTMs) of plants are similar to those of humans, slight differences can affect quality, safety and efficacy of PMPs (Walsh and Jefferis, Nat. Biotechnol., 24:1241-1252, 2006). One of the most common PTMs in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalyzed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human PTMs. Moreover, in plants the resulting hydroxyprolines are the anchor for O-glycosylation which again differs from human O-glycosylation.
Plant-based systems are gaining acceptance as alternative production platforms for recombinant biopharmaceuticals (Paul and Ma, Biotechnol. Appl. Biochem., 58:58-67, 2011). With regard to slight differences in post-translational modifications between humans and plants considerable progress was achieved in the humanization of Asparagin (N)-linked glycosylation of PMPs (Karnoup et al., Glycobiology, 15:965-981, 2005; Pinkhasov et al., Plant Biotechnol. J., 9:991-1001, 2011; Weise et al., Plant Biotechnol. J., 5:389-401, 2007, Cox et al., Nat. Biotechnol., 24:1591-1597, 2006). The attachment of immunogenic plant-specific β1,2-xylose and α1,3-fucose residues to the core N-glycan was abolished in different plant systems (Cox et al., Nat. Biotechnol., 24:1591-1597, 2006; Koprivova et al., Plant Biotechnol. J., 2:517-523, 2004; Strasser et al., FEBS Lett., 561:132-136, 2004; Sourrouille et al., Plant Biotechnol. J., 6:702-721, 2008). In addition, the elimination of Lewis A epitopes on N-glycans of rhEPO was reported recently (Parsons et al., Plant Biotechnol. J., 10:851-861, 2012). Further humanization of the N-glycosylation on PMPs was achieved by expression of the human β1,4 galactosyltransferase (Bakker et al., Proc. Natl. Acad. Sci. U.S.A, 103:7577-7582, 2006; Huether et al., Plant Biol. (Stuttg.), 7:292-299, 2005) and additional heterologous enzymes necessary for engineering sialylation (Castilho et al., J. Biol. Chem., 285:15923-15930, 2010). Despite this progress in humanizing N-glycosylation, differences in O-glycosylation can affect product quality. Plant O-glycosylation differs explicitly from the typical human mucin-type O-glycosylation (Gomord et al., Plant Biotechnol. J., 8:564-587, 2010) and induces antibody formation in mammals (Leonard et al., J. Biol. Chem., 280:7932-7940, 2005; Yates et al., Glycobiology, 6:131-139, 1996). Immunogenicity of biopharmaceuticals may result in reduced product efficacy and is a potential risk for the patients (Schellekens, Nat. Rev. Drug Discov., 1:457-462, 2002). Such adverse effects hamper the broad use of plants as production hosts for biopharmaceuticals. In plants, the main anchor for O-glycosylation is 4-trans-hydroxyproline (Hyp) (Kieliszewski, Phytochemistry, 57:319-323, 2001), while no further modification of Hyp occurs in mammals (Gorres and Raines, Crit. Rev. Biochem. Mol. Biol., 45:106-124, 2010). Although Hyp is always synthesized posttranslationally by prolyl-4-hydroxylases (P4Hs) via hydroxylation of the γ carbon of proline, recognition sites differ between mammals and plants (Gorres and Raines, Crit. Rev. Biochem. Mol. Biol., 45:106-124, 2010). Hyp is an important structural component of plant cell walls and of the extracellular matrix of animals and humans. Here, Hyp plays a key role in stabilizing the structure of collagen, one of the most abundant proteins in mammals, where the second proline of the tripeptide PPG is usually hydroxylated by collagen P4Hs. In plants, Hyp residues are the attachment sites for O-glycosylation in hydroxyproline-rich glycoproteins (HRGPs), the most abundant proteins in the plant extracellular matrix and cell wall. HRGPs include extensins, proline-rich glycoproteins and arabinogalactan proteins (AGPs) (Kieliszewski, Phytochemistry, 57:319-323, 2001; Kieliszewski and Lamport, Plant J., 5:157-172, 1994; Shpak et al., J. Biol. Chem., 276:11272-11278, 2001). Prolyl-hydroxylation and subsequent glycosylation of plant cell wall proteins is of major importance for growth, differentiation, development and stress adaption (Velasquez et al., Science, 332:1401-1403, 2011; Lamport et al., New Phytol., 169:479-492, 2006).
The target motifs for Hyp-anchored O-glycosylation in plants, so-called glycomodules, were defined and validated (Kieliszewski and Lamport, Plant J., 5:157-172, 1994; Shpak et al., J. Biol. Chem., 276:11272-11278, 2001). From these, the consensus motif [A/S/T/V]-P(1,4)-X(0,10)-[A/S/T/V]-P(1,4) (where X can be any amino acid) was derived for predicting prolyl-hydroxylation in plants (Gomord et al., Plant Biotechnol. J., 8:564-587, 2010). According to in silico analysis of the human proteome, approximately 30% of all proteins contain this motif, and are thus candidates for non-human prolyl-hydroxylation and subsequent O-glycosylation when expressed in plants (Gomord et al., Plant Biotechnol. J., 8:564-587, 2010). Consequently, adverse plant-typical prolyl-hydroxylation and even arabinosylation of PMPs was reported (Karnoup et al., Glycobiology, 15:965-981, 2005; Pinkhasov et al., Plant Biotechnol. J., 9:991-1001, 2011; Weise et al., Plant Biotechnol. J., 5:389-401, 2007). On the other hand, the artificial introduction of Hyp-O-glycosylation in PMPs was suggested as an alternative to PEGylation to increase the serum half-life of biopharmaceuticals (Xu et al., Biotechnol. Bioeng., 97:997-1008, 2007; US patent application 20060026719). However, non-human prolyl-hydroxylation does not only alter the native sequence of the protein, but also serves as anchor for O-glycans, which in turn may be immunogenic. Thus, the elimination of the anchor Hyp is the only safe way to avoid adverse O-glycosylation in PMPs.
The three documents EP 2 360 261 A1, Xu et al. (BMC Biotechnol, 11:69, 2011) and Stein et al. (Biomacromolecules, 10:2640-2645) each deal with the production of collagen in different plant systems (e. g. maize, tobacco). Mammalian- or human-specific prolyl hydroxylation is achieved by expression of exogenous mammalian/human prolyl 4 hydroxylase. Thus, the disclosed methods in all three documents require expression of exogenous mammalian/human prolyl 4 hydroxylase.
Among plants, the moss Physcomitrella patens offers the unique possibility for precise and targeted genetic engineering via homologous recombination (e.g. Strepp et al., Proc. Natl. Acad. Sci. U.S.A, 95:4368-4373, 1998; Koprivova et al., Plant Biotechnol. J., 2:517-523, 2004). Further, several PMPs have been produced in the moss bioreactor, including rhEPO (Decker and Reski, Plant Cell Rep., 31:453-460, 2012), the leading biopharmaceutical world-wide. Its market turnover is over 10 billion Euros per year. EPO is a highly glycosylated peptide hormone stimulating erythropoiesis. Recombinant hEPO produced in CHO (Chinese hamster ovary) cells is used for prevention or treatment of anemia in nephrology and oncology patients, and can be abused for illegal doping activities. A glyco-engineered version of EPO (asialo-EPO) has no hematopoietic activity but can serve as a safe drug with neuro- and tissue-protective functions after stroke and additional hypoxia stress (Erbayraktar et al., Proc. Natl. Acad. Sci. U.S.A, 100:6741-6746, 2003). Production of correctly N-glycosylated asialo-EPO in the moss bioreactor was reported recently (Parsons et al., Plant Biotechnol. J., 10:851-861, 2012). However, plant-derived rhEPO is hydroxylated within the motif SPP (147-149) (Weise et al., Plant Biotechnol. J., 5:389-401, 2007) and thus may have adverse effects on patients.
Weise et al. (Plant Biotechnol. J., 5:389-401, 2007) and Parsons et al. (Plant Biotechnol. J., 10:851-861, 2012) both deal with the production of rhEPO in moss and with the modulation of the glycosylation pattern of N-glycans by targeting the plant-specific fucosyl-/xylosyl-/galactosyltransferases. Thereby, immunogenic fucoses/xyloses/galactoses on the N-glycans are removed. Both documents do not address O-glycosylation because prolyl hydroxylation (as an anchor for O-glycosylation) is not subject of these disclosures.
The hydroxylation of prolines of a recombinant human protein produced in a plant-based system cannot be obviated so far. It only becomes apparent after production of a recombinant human protein if its prolines are hydroxylated and if non-human or plant specific O-glycosylation is present.
It is an object of the present disclosure to provide a method for the production of a recombinant protein using a plant-based system. It is also an object of the present disclosure to provide a recombinant protein, which has been produced in a plant-based system without the need to introduce exogenous prolyl-4-hydroxylase genes into the system, wherein the recombinant protein does not comprise any non-human prolyl hydroxylation. It is further an object of the present disclosure to provide a plant-based system used for the production of such a recombinant protein and to provide a use of such a recombinant protein.